MYC-containing double minutes in hematologic malignancies: evidence in favor of the episome model and exclusion of MYC as the target gene

Double minutes (dmin)—circular, extra-chromosomal amplifications of specific acentric DNA fragments—are relatively frequent in malignant disorders, particularly in solid tumors. In acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), dmin are observed in ∼1% of the cases. Most of them consist of an amplified segment from chromosome band 8q24, always including the MYC gene. Besides this information, little is known about their internal structure. We have characterized in detail the genomic organization of 32 AML and two MDS cases with MYC-containing dmin. The minimally amplified region was shown to be 4.26 Mb in size, harboring five known genes, with the proximal and the distal amplicon breakpoints clustering in two regions of ∼500 and 600 kb, respectively. Interestingly, in 23 (68%) of the studied cases, the amplified region was deleted in one of the chromosome 8 homologs at 8q24, suggesting excision of a DNA segment from the original chromosomal location according to the ‘episome model'. In one case, sequencing of both the dmin and del(8q) junctions was achieved and provided definitive evidence in favor of the episome model for the formation of dmin. Expression status of the TRIB1 and MYC genes, encompassed by the minimally amplified region, was assessed by northern blot analysis. The TRIB1 gene was found over-expressed in only a subset of the AML/MDS cases, whereas MYC, contrary to expectations, was always silent. The present study, therefore, strongly suggests that MYC is not the target gene of the 8q24 amplification

NPM1 Deletion Is Associated with Gross Chromosomal Rearrangements in Leukemia

BACKGROUND: NPM1 gene at chromosome 5q35 is involved in recurrent translocations in leukemia and lymphoma. It also undergoes mutations in 60% of adult acute myeloid leukemia (AML) cases with normal karyotype. The incidence and significance of NPM1 deletion in human leukemia have not been elucidated. METHODOLOGY AND PRINCIPAL FINDINGS: Bone marrow samples from 145 patients with myelodysplastic syndromes (MDS) and AML were included in this study. Cytogenetically 43 cases had isolated 5q-, 84 cases had 5q- plus other changes and 18 cases had complex karyotype without 5q deletion. FISH and direct sequencing investigated the NPM1 gene. NPM1 deletion was an uncommon event in the "5q- syndrome" but occurred in over 40% of cases with high risk MDS/AML with complex karyotypes and 5q loss. It originated from large 5q chromosome deletions. Simultaneous exon 12 mutations were never found. NPM1 gene status was related to the pattern of complex cytogenetic aberrations. NPM1 haploinsufficiency was significantly associated with monosomies (p<0.001) and gross chromosomal rearrangements, i.e., markers, rings, and double minutes (p<0.001), while NPM1 disomy was associated with structural changes (p=0.013). Interestingly, in complex karyotypes with 5q- TP53 deletion and/or mutations are not specifically associated with NPM1 deletion. CONCLUSIONS AND SIGNIFICANCE: NPM1/5q35 deletion is a consistent event in MDS/AML with a 5q-/-5 in complex karyotypes. NPM1 deletion and NPM1 exon 12 mutations appear to be mutually exclusive and are associated with two distinct cytogenetic subsets of MDS and AML

Detection of Chromosomal Abnormalities in Chronic Lymphocytic Leukemia Increased by Interphase Fluorescence In Situ Hybridization in TPA-Stimulated Peripheral Blood Cells.

Abstract Chromosomal abnormalities, namely deletion 11q-,13q-, 17p- and trisomy 12, have prognostic significance for patients with chronic lymphocytic leukemia (CLL). Several studies have demonstrated that the interphase fluorescence in situ hybridization technique (I-FISH) in CLL identifies such genomic aberrations in a higher frequency than classical karyotyping, including stimulated cultures using B-cell specific mitogens. However, there appears to be no information in the literature comparing I-FISH on non-cultured and cultured cells in CLL. A total of 56 samples from 49 patients with CLL were studied using commercially available probes for chromosomes 11q22.3(ATM), 13q14(13S272), 17p13(p53) and 12 centromere(D12Z3). We compared the results obtained by I-FISH-PBMC and those by interphase fluorescence in situ hybridization on TPA-stimulated peripheral blood cells (I-FISH-TPA) used for conventional cytogenetics in order to evaluate the usefulness of I-FISH-TPA. The proportion of abnormal nuclei obtained with the I-FISH-TPA was higher than that found with I-FISH-PBMC (P&lt;0.001). Consequently, 15 cases with a negative or borderline result by I-FISH-PBMC became positive by I-FISH-TPA for deletion 11q- (n=2), 13q- (n= 9) and trisomy 12 (n=4). In all but one of these, chromosomal abnormalities were reconfirmed by either metaphase-FISH or conventional G-banding. Disease detection thus increased from 51% with I-FISH-PBMC to 78% with I-FISH-TPA. Interestingly, all 15 cases which reached the diagnostic thresholds for deletion 11q-,13q- and trisomy 12 had a slight lymphocytosis. An absolute lymphocyte count &lt;8.7×109/l was found to be the critical threshold (P=0.037) below which I-FISH-TPA should be performed rather than I-FISH-PBMC. We have shown that I-FISH-TPA can not only detect a higher proportion of abnormal interphase nuclei but can also identify abnormal CLL cases which may be overlooked by I-FISH-PBMC, especially those with low absolute lymphocyte counts. I-FISH-TPA is thus a reliable technique for clinical diagnostics in CLL.</jats:p

The Use of Tetradenoylphorbol Acetate Stimulated Peripheral Blood Cells for Interphase Fluorescence In-Situ Hybridization Analysis May Enhance Its Prognostic Value for Patients with Chronic Lymphocytic Leukemia

Abstract Chronic lymphocytic leukemia (CLL) is a mature B-cell malignancy characterized by a variable clinical course. Genomic aberrations, as identified by interphase fluorescent in situ hybridization (I-FISH), have a remarkable predictive power in terms of response to therapy, time to first treatment and overall survival of these patients. I-FISH studies can be performed either on unstimulated peripheral blood mononuclear cells (I-FISH-PBMC) or on tetradecanoylphorbol acetate (TPA) stimulated peripheral blood cells (I-FISH-TPA). We have previously observed that I-FISH-TPA could identify genomic abnormaties that might be overlooked with I-FISH-PBMC. The aim of the study was to evaluate whether this increased detection of genomic abnormalities, as identified by I-FISH-TPA, was clinically relevant for a group of consecutive CLL patients. Blood samples from 47 CLL patients were stimulated with TPA and cultured in RPMI medium supplemented with fetal calf serum. Peripheral blood mononuclear cells were isolated using density-gradient centrifugation and fixed according to standard methods. I-FISH was performed on both TPA stimulated and unstimulated cells using 11q22.3 (ATM), 13q14 (13S272), 17p13.1 (p53), and centromeric 12 (D12Z3) probes. 200 nuclei were evaluated for each probe, and cut-off points were set at 6%, 7%, 5% and 2% for del(11q), del(13q), del(17p) and trisomy 12, respectively. Metaphase FISH and conventional cytogenetics were also performed in selected cases. For all patients, chemotherapy was initiated according to Cheson criteria and treatment-free and overall survival curves were plotted using SPSS software. Following a modified version of Dohner’s hierarchical model, patients were divided in those with del(17p) and/or del(11q) and those with other or no genomic abnormalities. Fourteen cases with negative or bordeline results with I-FISH-PBMC became positive with I-FISH-TPA for del(11q) (2 cases), del(13q) (9 cases) and trisomy 12 (3 cases). In all but one patient, either conventional karyotyping or metaphase FISH confirmed these abnormalities. I-FISH-TPA provided a better prediction of treatment-free interval compared to I-FISH-PBMC (P= 0.002 vs 0.019, see Figures 1 and 2). In particular, two patients with no cytogenetic abnormalities detected by I-FISH-PBMC required chemotherapy 3 and 11 months after diagnosis, more in keeping with the presence of del(11q) found using I-FISHTPA. Furthermore, I-FISH-TPA also improved the overall survival prediction compared to I-FISH-PBMC (P= 0.036 vs 0.042). In summary, I-FISH-TPA increased the detection rate and had an improved prognostic value compared to I-FISH-PBMC. Further studies with larger numbers of patients are warranted. Figure Figure Figure Figure</jats:p

Treatment of Chronic Myeloid Leukemia with Imatinib. A Single Centre Experience

Abstract With the introduction of imatinib (IM) as first-line therapy for chronic myeloid leukemia (CML) as well as in advanced phases of the disease, the long-term outcome of this hematologic malignancy has dramatically changed.. The aim of this study has been to analyse the experience of our centre with IM therapy in CML patients, focusing on the cytogenetic and molecular responses according to the phase of the disease in which IM was given, as well as the treatment failures and the results of the mutational analysis performed in these situations. Between January 2000 and March 2008, 77 patients with CML [47 men and 30 women, median age 49 years (range, 18–78)] were treated with IM. 33 of them received IM at a dose of 400 mg qd as first-line treatment in de novo chronic-phase (CP), 32 patients received it in late chronic-phase [time from diagnosis to IM, 51 (12–157) months]. Of these 32 patients in late CP, all of them received prior therapy with interferon-alpha, 10 patients received an autologous transplant and 3 of them an allogeneic transplant. Five patients were initially treated with IM 600mg qd because of an accelerate-phase (AP) and the remaining 6 patients were treated with IM 800mg qd because of a blastic-phase (BP). De novo CP Late CP AP BC CHR(%) 32 (96) 28 (87) 4 (80) 3 (50) Time to CHR in months [median (range)] 1,8 (0,5–6) 1 (3–10) 2 (0,5-4) 3 (1–5) CCR(%) 30 (90) 22 (68) 3 (60) 1 (17) Tims to CCR in months [median (range)] 11 (6–63) 10 (6–36) 12 (7–18) — MMoIR(%) 20 (60) 15 (47) 1 (20) 1 (17) Time to MMoIR in months [median (range)] 18 (6–68) 22 (6–58) — — CMoIR(%) 0 0 0 0 Time to CMoIR in months [median (range)] — — — — Relapses (%) 5 (15) 9 (28) 4 (80) 5 (87) Mutational analysis (n, %) 4 (80) 6 (66) 3 (75) 3 (50)     Positive 2 (50) 2 (33) 1 (33) 0     Negative 2 (50) 4 (67) 2 (67) 3 (100) Second-line treatment     Dasatinib 1 3 0 1     Allo-SCT 1 2 0 1     Others — 3 3 — Follow-up from diagnosis 41 (11–90) 103 (34–215) 107 (37–185) 74 (13–158) Follow-up from beginning of IM 43 (11–97) 52 (6–87) 52 (13–83) 22 (1–88)     Alive 31(94) 22(69) 3(60) 2(33)     Death 2(6) 10(31) 2(40) 4(67) The results of our centre confirm the ones presented in prospective analysis. There is a small percentage of patients that relapse or progress under IM therapy, and this percentage is significantly higher in patients treated in advanced phases. Mutational analysis is relevant in up 50% of the patients studied, allowing changes in therapy according to the obtained results.</jats:p

Improved Response to 5-Azacitidine in Patients with Primary Compared to Secondary AML, Particularly If NPM1 Mutations Are Present,

Abstract Abstract 3562 5-azacitidine (5-aza) is a widely-used agent for treatment of myelodysplastic syndromes (MDS). In contrast, data on its use in AML are far more limited and, to date, little is known on the clinical or biological markers predicting the response of AML to this drug. We retrospectively analysed a series of patients with AML who received 5-aza due to old age, poor condition or refractoriness to standard treatment, and investigated the potential association of a variety of genetic markers with response and survival. PATIENTS AND METHODS: From 2006 to 2011, 38 patients (23 males, 15 females) diagnosed as AML according to the WHO criteria1 received treatment with 5-aza (75 mg/m2 SQ daily, 5–7 days) at our institution as on-label or compassionate use program. Twenty-four patients (median age 68 years, range 37–85) had primary AML (pAML). Most of them (n=18) had failed to prior standard AML treatment: one line in 12 cases, 2 lines in 5 (including stem cell transplant [SCT] in 2 cases), and 3 lines in one. Fourteen additional patients (median age 68.3; range 32–88) had secondary AML (sAML); among them 8 previously treated: 6 patients had received one line of standard treatment before 5-aza, 1 patient 2 lines, and 1 patient 3 different lines. Twenty-two pAML and 14 sAML cases were screened for the presence of karyotype abnormalities. Screening for gene mutations [FLT3 internal tandem duplication (FLT3/ITD, nucleophosmin-1 gene mutation (NPM1), MLL partial tandem duplication, CCAAT/enhancer binding protein alpha mutation (CEBPA), AML1-ETO and CBFB-MYH11] was performed prior to starting 5-aza whenever possible. The response to treatment was usually assessed between the 4th and 6th courses, and classified according to WHO criteria. RESULTS: Patients received a median 7 courses (range 1–19) of standard-dose 5-aza. Within the pAML group, 13 patients responded [8 complete responses (CR) and 5 partial responses (PR)], and 4 patients with sAML responded (2 CR and 2 PR). Two patients in the pAML group and 1 patient with sAML subsequently received an SCT. After a median 218 days (10–792) follow-up in the pAML group, and 246 (19–995) in the sAML group, 66.7% patients with pAML and 28.6% with sAML were alive. Overall, no significant differences were seen in the rate of patients achieving CR or PR between the pAML and the sAML groups; despite this, the estimate 2-year survival was significantly higher in patients with pAML (p=0.01). Prior exposure to chemotherapy or other forms of treatment had no impact on response or survival. An abnormal karyotype (present in 12 pAML and 8 sAML patients) did not influence the response to treatment. All mutation analysis yielded negative results in sAML cases. In pAML cases, 3/20 were positive for FLT3/ITD, 5/19 for NPM1 and 1/19 for MLL. Most interestingly, NPM1 mutations in the absence of FLT3/ITD, were significantly (p=0.05) associated with improved response rate (5/5) in patients with pAML with no deaths being observed in this group. DISCUSION: Our results show a higher probability of survival after 5-aza treatment in patients with pAML compared to sAML. The presence of NPM1 mutations appeared to identify a subset of pAML patients that, despite having failed first line treatment, may be particularly sensitive to this therapy. If confirmed in larger series, this finding would be of the utmost interest for offering these patients a low toxicity rescue therapy or a maintenance strategy with his agent. Disclosures: No relevant conflicts of interest to declare. </jats:sec

Correlation between WT1 Levels and Chimeric Transcripts in Core Binding Factor AML: Discrepancies Are Not Uncommon AML1-ETO Leukemias.

Abstract The Wilms tumor gene (WT1) was identified as a tumor suppressor gene coding for a zinc-finger transcription factor. It has been demonstrated that WT1 is overexpressed in acute leukemias and detection of increased levels of WT1 transcripts mainly in peripheral blood, has been associated with clonal growth and relapses. Most AML patients do not have a suitable molecular marker for minimal residual disease (MRD) monitoring. WT1 quantification seems to be an attractive strategy of universal leukemia follow-up. Bone marrow samples from 46 core binding factor (CBF) AML patients were tested for WT1 expression in parallel to AMLI/ETO and CBFb/MYH11quantification. Total RNA was purified using the Trizol reagent (Invitrogen). Chimeric detection was performed following the BIOMED recommendations. The WT-1 mRNA expression was measured by real-time quantitative polymerase chain reaction (RQ-PCR) in an ABI7700 genetic analyzer (Applied Biosystems, Foster City). For WT1 copy number titration the IPSOGEN plasmid was employed (Ipsogen, Marseilles). One hundred and fifty bone marrow (BM) samples from AML1-ETO patients (23 samples at diagnosis and 127 during follow-up) and 195 from CBFbeta-MYH11 cases (21 samples at diagnosis and 174 during follow-up) were included in the study. Bone marrow samples from 6 healthy donors were used to establish the highest acceptable value of WT1 copy number (80 copies). The WT1 expression was significantly increased (up to 3 orders) in bone marrow samples of AML patients at diagnosis compared to BM samples of healthy donors (P &lt; 0.0001). Relapses were observed exclusively in the CBFbeta-MYH11 group and were always preceded by rising amounts of WT1 levels. A good concordance between WT1 levels (80 copies) and prognostically relevant chimeric trancript copy number (1 copy) was detected in 96.23% of the samples. Discrepancies between WT1 and specific fusion genes were observed in 11 AML1-ETO samples and in two CBFbeta-MYH11 cases. In ten follow up AML1-ETO samples with copy values of 1 or less a WT1 result exceeding the 80 threshold was detected. This discordant result was found in a single CBFbeta-MYH11 sample. Our findings suggest that WT1 is a reliable MRD marker in CBFbeta-MYH11 AML. It remains to be investigated the meaning of high titers of WT1 in AML1-ETO acute leukemias.</jats:p

Structural alterations in chronic lymphocytic leukaemia. Cytogenetic and FISH analysis

In this study, we described cytogenetics and fluorescence in situ hybridization (FISH) analysis performed in chronic lymphocytic leukaemia (CLL) patients with structural alterations. Results were correlated with clinical characteristics. A total of 38 CLL patients: 16 cases with complex and 22 with simple karyotypes were studied. For comparison of clinical parameters, a control group of 78 CLL patients with normal karyotype and without FISH genomic alterations were also evaluated. We found 38 structural abnormalities not previously described in the literature, 28 (74%) of them were translocations. In cases with complex karyotypes, chromosomes 6, 8 and 13 were the most frequently involved in new alterations (nine each), followed by chromosomes 12, 14 and 15 (six each). Chromosome 8p was particularly involved in losses, being 8p21-pter the commonest region of overlap. Cases with simple karyotypes, showed del(6q) as the most frequent alteration (39%). Del(9)(q11) was recurrent in our series. Analysis of clinical parameters showed significant differences in white blood count (p=0.005) and platelet count (p=0.015) between patients with structural alterations and the control group. In addition, patients with structural alterations had a significantly shorter time to first treatment (TFT) (29months) than the control group (69months) (p=0.037). Cases with complex karyotypes had a lower proportion of patients in Rai 0 clinical stage (15.4% vs 75%) (p=0.005) and higher β2 microglobulin levels (3.3 vs 2.5μg/mL) (p=0.037) than those with simple karyotypes. Furthermore, a shorter TFT (13months) and overall survival (56months) in the complex karyotypes group compared with controls (69 and 144months, respectively) (p=0.015 and p=0.005, respectively) were also found. Our results support the importance of cytogenetic analysis for clinical outcome in CLL and suggest that the diversity of genomic alterations is much greater than previously appreciated. © 2012 John Wiley & Sons, Ltd.Fil: Travella, Ana Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Ripollés, Lorena. Universitat Autònoma de Barcelona; EspañaFil: Aventin, Anna. Hospital de Sant Pau; EspañaFil: Rodríguez, Andrea. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Bezares, Raimundo F.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Dr. Teodoro Álvarez"; ArgentinaFil: Caballín, María R.. Universitat Autònoma de Barcelona; EspañaFil: Slavutsky, Irma Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

Uniparental Disomy May Be Associated with NPM Mutations in AML with a Normal Karyotype.

Abstract Acute myeloid leukemia (AML) is a heterogeneous group of neoplastic disorders characterized by an abnormal proliferation of the myeloid precursors and a maturation block. A large proportion of AML cases have either a normal karyotype or non-recurrent chromosomal alterations. Underlying genetic lesions of some of these cases have been characterized with the discovery of MLL-internal tandem duplications, activating FLT3 mutations and NPM mutations. Loss of heterozygosity (LOH) derives from the loss of one of the two alleles at a given locus and can be a sign of inactivation of tumor-suppressor genes. We performed a high-resolution genotype analysis on DNA obtained from 19 AML patients with a normal karyotype, both at diagnosis and in samples obtained in complete remission(assessed by multiparametric flow cytometry) using the 10K SNP Array (Affymetrix). Both LOH and copy number analysis, as well as visualization of these analysis were performed by means of the dChip software (M. Lin et al., Bioinformatics (2004), 20:1233–40). A mean call rate of 96.8%. SNP array-based LOH analysis revealed that 4 patients presented large regions of homozygosity at diagnosis which were absent from samples in complete remission. In all four patients copy number analysis indicated no gross chromosomal losses or gains, as was confirmed by conventional cytogenetic analysis. Therefore, it can concluded that the LOH observed in these four patients was due to the presence of uniparental disomy. Simultaneous analysis of FLT-3 internal tandem duplications (FLT-3/ITD), FLT3- D835 mutations, NPM mutations and MLL rearrangements was performed using conventional molecular methods. Two of these patients (UPN2 and UPN12) had FLT-3/ITD in association with NPM mutations. UPN4 had a mutated form of NPM whereas in patient UPN16 FLT-3 and NPM genes were in the germ line configuration. All four cases were negative for MLL rearrangements and FLT-3-D835 mutations. These results suggest that NPM and FLT3 mutations may be associated with acquired somatic recombinations. It remains to be investigated whether there are loci preferentially involved by these events. Uniparental disomy and genetic lesions in normal karyotype AML Patient LOH FLT3 NPM D835 MLL UPN2 13q Mutated Mutated Germ line Germ line UPN4 6pter-p12.212q13.12-qter Germ line Mutated Germ line Germ line UPN12 2p Mutated Mutated Germ line Germ line UPN16 complex Germ line Germ line Germ line Germ line</jats:p